The present invention relates to a determination method in particular to a method, based on immunoassay, for the determination of water treatment chemicals in aqueous media, and to novel antibodies and hybridomas useful in the new method.
The majority of natural waters, and aqueous systems in general, contain dissolved salts of metals such as calcium, magnesium, barium and strontium. When the natural water or aqueous system is heated, the dissolved salts may be converted to insoluble salts, and thereupon deposited as scale on any heat transfer surfaces in contact with the water or aqueous system. Insoluble salt scale may be formed even when the water or aqueous system is merely concentrated, without being heated.
Such precipitation and scale deposition are troublesome and can result in an increase in the costs required to maintain aqueous systems in good working order. Among the problems caused by scale deposits are obstruction of fluid flow, impedance of heat transfer, wear of metal parts, shortening of equipment life, localised corrosion attack, poor corrosion inhibitor performance and unscheduled equipment shutdown. These problems can arise, e.g. in any circulating water system such as those used in oil drilling wells, steam power plants, water desalination plants, reverse osmosis equipment, heat exchange equipment and equipment concerned with the transport of products and by-products in aqueous media, e.g. fly-ash formed during the combustion of coal, in the production of electricity
A number of additives, notably polycarboxylates, have been provided as effective scale inhibitors for addition to aqueous systems.
Likewise, natural waters and aqueous systems are corrosive towards metals which are in operational contact with them. Consequently, such aqueous systems must be treated with a corrosion inhibitor, e.g. a phosphonate, in order to prevent deterioration of such metals, e.g. pipelines.
Although water treatment chemicals can be effective at very low concentrations, a certain minimum concentration must be maintained if the aqueous system is to operate trouble-free. With the passage of time, loss of the water treatment chemical from the system occurs and replenishment is necessary to avoid the above-mentioned operational problems. On the other hand, use of excess of water treatment chemical increases operational costs. The need to balance treatment, chemical effectiveness and cost has led, therefore, to the development of methods and devices for monitoring the level of water treatment chemicals in aqueous systems.
For example, colourimetric methods are available for the determination of scale inhibitors, e.g. polycarboxylates. Colorimetric methods, however, have the disadvantage that they are subject to interference from extraneous materials. In oil field applications, for instance, interference arises mainly from iron and oil-derived organic materials.
In an attempt to overcome this interference problem, a sample-preparation (pretreatment) cartridge maybe employed, in which interfering species are removed and the water treatment chemical is concentrated. Unfortunately, however, such techniques can result in loss of the water treatment chemical being determined due to competition from the organics for adsorption sites on the cartridge. Such methods are time consuming, lack robustness and the required sensitivity (limits of detection only 1-2 ppm). In addition they require a certain amount of expertise in order to be used effectively to conduct the required determination.
More recently, immunological methods have been developed for the determination of organic compounds.
Immunological methods for determining proteins, cells, hormones, vitamins, drugs and mycotoxins etc. have been known for many years, and have been widely reported in the literature. In such methods, an animal, often a mouse or rabbit, is immunized either with an analyte or a protein-analyte conjugate. The antibodies produced by the animal are then used, in the form of an immunoassay, to determine the analyte. These methods are based upon the specific reaction between the analyte and the antibody.
The immunoassays which have been reported in the literature incorporate antibodies that have been raised to natural molecules. Recently, however, EP 260829A, has disclosed novel mono- and polyclonal antibodies which are reactive with chlorinated phenols, especially pentachlorophenol. The antibodies can then be used to identify and assay pentachlorophenol, which is widely used as a pesticide and preservative.
We have now succeeded in applying an immunoassay method to the detection of water treatment polymers in aqueous solution, to provide a determination method which is sensitive, specific, rapid, robust and which can be operated by relatively inexperienced personnelxe2x80x94this has not been achieved by such methodology before the present application.
It is surprising that an antibody can be raised effectively to molecules which are polydisperse i.e. having differing molecular weights which vary considerably in size and shape. The competitive assay results demonstrate that the antibodies are raised to the core active centre of the molecules i.e. a moiety which is present in every molecule in the product although the number of repeating monomer units can vary.
Accordingly, the present invention provides a method for determining the presence and/or concentration of a water treatment polymer in an aqueous sample, comprising the production of polyclonal or monoclonal antibody to the water treatment polymer; and using the antibody so produced as a reagent in an immunoassay conducted on the aqueous sample.
The present invention also provides a method for determining the presence and/or concentration of a water treatment polymer in an aqueous sample, comprising an effective amount of a monoclonal antibody or polyclonal antibody which has been raised to the water treatment polymer, in association with an acceptable carrier.
Preferred water treatment polymers, for determination in the process of the present invention, are phosphorus acid containing carboxylic acid telomers having the formula I: 
or salts thereof, in which Rxe2x80x3 is hydrogen, methyl or ethyl, R is hydrogen, C1-C18 alkyl, C5-C12 cycloalkyl, aryl, aralkyl, a residue of formula: 
in which Rxe2x80x3 has its previous significance and the sum of m and n is an integer of at most 100, or R is a residue xe2x80x94OX in which X is hydrogen or C1-C4 alkyl, and R1 is a residue xe2x80x94OX in which X has its previous significance.
The telomers of formula I, and their production are described in more detail in U.S. Pat. No. 4,046,707.
Particularly preferred telomers of formula I are those having the formula IA: 
in which the sum of mxe2x80x2 and nxe2x80x2 is an integer ranging from 4 to 32, especially, 15 to 20.
Other preferred water treatment polymers, for determination in the process of the present invention are hydrolyzed terpolymers of maleic anhydride with other monomers the molar ratio of maleic anhydride to the other monomers ranging from 2.5:1 to 100:1 and the molecular weight of the terpolymer being below 1000. Such terpolymers are described in U.S. Pat. No. 4,126,549.
Preferred ratios of monomers in the terpolymer are in the range of 2xc2xd-3xc2xd:1 of maleic anhydride to other monomers. Preferred other monomers are vinyl acetate acid and ethyl acrylate.
These ratios are those used in the preparation of the cotelomer of formula II and are not necessarily the ratios to be found in the final cotelomer.
Other examples of preferred water treatment molecules include other polyacrylic acid polymers; copolymers of acrylic acid and acrylamidomethylpropane sulphonic acid (AMPS); copolymers of acrylic acid and vinyl acetate; polymaleic acid; hydrolysed polymaleic acid; terpolymers of maleic acid, ethyl acrylate and vinyl acetate; copolymers of acrylic acid and maleic anhydride; copolymers of maleic acid and sodium allyl sulphonate; and copolymers of maleic anhydride and sulphonated styrene and vinyl sulphonic acid telomers.
With respect to aqueous systems in which water treatment polymers to be determined may be present, of particular interest are the aqueous systems employed in cooling water plant steam generating plant, sea-water evaporators, reverse osmosis equipment, paper manufacturing equipment, sugar evaporator equipment, soil irrigation plant, hydrostatic cookers, gas scrubbing systems, closed circuit heating systems, aqueous-based refrigeration systems and down-well systems.
The antibody used in the method and composition of the present invention may be produced by known techniques.
For the production of polyclonal antibodies which are reactive with a particular water treatment polymer, firstly an immunogenic conjugate of the polymer and a macromolecule carrier may be produced; an animal may then be immunized with the conjugate, the polymer alone, adjuvant or a discrete mixture of each; blood may be removed from the animal and the serum separated from the blood; and finally the polyclonal antibodies may be recovered from the serum.
It may be preferred, however, to use monoclonal antibodies, which are reactive with specific epitopes on the water treatment polymer, in the method and composition of the present invention, especially in view of their superior specificity for a particular polymer. Monoclonal antibodies may be obtained by the technique first described by Kohler and Milstein, Nature, 265:495 (1975). This technique comprises providing an immunogenic form of the specific water treatment polymer, immunizing an animal with such; obtaining antibody-producing cells from the animal; fusing the cells so obtained with myeloma cells to produce hybridomas; selecting from the hybridomas a hybridoma which produces an antibody which reacts with the specific water treatment polymer, and then isolating the monoclonal antibody from the selected hybridoma
Water treatment polymers generally have low molecular weights and do not, per se, induce the production of antibodies. They can be used as a hapten, however, in combination with a higher molecular weight, immunogenic carrier, such as a protein, using e.g. the technique disclosed by Albro et al. Toxicol Appl. Pharmacol 50, 137-146 (1979).
The conjugate so obtained may then be used to immunize an animal host, by conventional techniques, e.g. inoculation. The animal host may be, e.g. a rabbit or a rodent such as a rat or mouse.
After the host animal has produced antibodies to the administered conjugate, polyclonal antibodies may be recovered from the animal by conventional techniques.
For example, blood may be removed from the animal and serum may be separated from the blood so removed. The desired antibodies may then be removed from the serum, e.g. by affinity purification or salt fractionation.
To produce monoclonal antibodies to the water treatment polymer, cells which produce antibodies may be recovered from the immunized animal. B lymphocytes removed from the animal""s spleen are preferred.
The removed cells are fused with myeloma cells to produce hybridomas, which are then separated, again using standard techniques such as cloning by limiting dilution.
Once the hybridomas have been separated a selection is made to ascertain those which produce antibodies to the specific water treatment polymer to be determined in the method of the present invention. The relevant specific hybridomas can then be isolated by known methods, and the relevant antibodies secreted from them by conventional techniques.
The following examples further illustrate the present invention.